Therapeutic application of small interfering RNA (siRNA) requires efficient and specific systemic delivery in vivo to target cells. Antibodies targeting internalizing cell surface epitopes are promising targeting agents for systemic delivery of siRNA. Recently, successful in vivo gene silencing has been achieved using several antibody fragments, demonstrating the potential of this approach. It is likely, however, that different antibodies will have varying abilities to act as targeting agents for siRNA, due to differences in binding epitopes, affinity, internalizing activity, and subcellular trafficking post-internalization. Thus, antibodies optimal for functional siRNA delivery may best be identified empirically using functional assays. Currently, no systematic evaluation of the targeting and delivery potential of a panel of antibodies has been done. In addition, no antibody-mediated siRNA delivery to prostate cancer has been studied. Combining phage antibody display and laser capture microdissection, we have previously identified a panel of internalizing single chain variable fragments (scFvs) that target clinically represented prostate cancer cell surface antigens. These scFvs were selected for their ability to bind to tumor cells in situ, and to mediate efficient and specific intracellular payload delivery, and thus are promising candidate for the delivery of siRNA. We propose to develop targeted in vivo siRNA delivery vehicles utilizing this panel of scFvs with the goal of developing nanosized targeted siRNA therapeutics against prostate cancer. Aim 1: To develop internalizing antibody-based nanosized siRNA delivery complexes and evaluate their ability to silence gene expression in vitro. We propose to modify these internalizing scFvs to impart them with siRNA binding functions. To enhance endosome escape, we propose to introduce an additional endosomolytic moiety to the targeting complex. We will evaluate siRNA delivery and gene silencing in vitro using these nanosized siRNA-targeting complexes. Aim 2: To achieve targeted systemic siRNA delivery in vivo to prostate cancer cells. Following the in vitro study, the most potent scFv/siRNA targeting complexes will be selected for in vivo study using prostate cancer xenografts. Following systemic administration, their ability to silence reporter genes as well as genes involved in prostate cancer growth and homeostasis will be evaluated in vivo. PUBLIC HEALTH RELEVANCE: We have previously identified a panel of phage antibody-derived;internalizing human monoclonal antibodies that target clinically represented prostate cancer cell surface antigens. We propose to develop nanosized targeted small interfering RNA (siRNA) delivery vehicles utilizing this panel of antibodies. This study will generate a panel of novel reagents for efficient and specific siRNA delivery in vivo to silence disease-causing genes, and will facilitate the development of nanosized siRNA-based prostate cancer therapeutics.